Purpose: To evaluate the antiamoebic properties of photo-activated chromophore for keratitis (PACK)-corneal cross-linking (CXL) (PACK-CXL), in combination with riboflavin (0.1 and 0.25%) or rose bengal (0.1 and 0.2%), for treatment of Acanthamoeba trophozoites and cysts.Materials and Methods: Cultures of Acanthamoeba castellanii were grown in a fluid medium at a concentration of 2.7x10(5) cell/ml. PACK-CXL was used on A. castellani cells in combination with either riboflavin (0.1 and 0.25%) or rose bengal (0.1 and 0.2%). Riboflavin-containing wells were irradiated with ultraviolet-A (UVA) light (365-nm wavelength). Rose bengal-containing wells were irradiated with green light (523-nm wavelength). A power density of 9mW/cm(2) for 10min and total irradiation dose of 5.4J/cm(2) was used for both riboflavin and rose bengal. After UVA and green light irradiation, cell viabilities were evaluated, and percentage of dead cells calculated.Results: No significant amoebicidal activity was observed following PACK-CXL/riboflavin at either concentration. PACK-CXL/rose bengal, however, was observed to be highly effective in eradicating Acanthamoeba cells at either concentration, with no significant difference observed between the two concentrations. The percentage of dead cells was 63% following treatment at either rose bengal concentration.Conclusion: PACK-CXL with rose bengal demonstrated pronounced antiamoebic activity against A.castellanii. Further in vitro and in vivo studies are needed to confirm this finding.